The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitroquinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 !g/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV < 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Amphibians are useful biological indicators because their sensitive skins and occurrence in aquatic and terrestrial habitats makes them vulnerable to environmental change (Blaustein and Wake, 1990; Lips, 1998). Studies using frogs as models to measure the environmental impact of xenobiotics have used mainly larval phases of biphasic frog species such as Anaxyrus americanus,Hyla versicolor, Lithobates catesbeianus,Lithobates clamitans and Lithobates pipiens (Berrill et al., 1994; Relyea, 2004a,b). Most Neotropical frogs have direct development and reproduce on land (Hedges et al., 2008). However, few studies have assessed the usefulness of these species as environmental bioindicators.

Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) is a direct-developing frog (Hedges et al., 2008) from the Lesser Antilles with recently established populations in several Caribbean islands (Kaiser, 1997), as well as in Central and South America (Ortega et al., 2001; Kaiser et al., 2002). As a consequence of its widespread distribution and certain life history features (reproductive flexibility and high environmental adaptability) (Ortega et al., 2005), E. jonhstonei has been considered a successful invasive species (Rödder, 2009). These attributes also suggest that this species could be a useful model for evaluating the genotoxicological impact of environmental xenobiotics such as pesticides.

DNA damage by environmental xenobiotics is frequently assessed by single cell gel electrophoresis (SCGE) or the Comet assay (Singh et al., 1988), which detects DNA strand breakage and alkali-labile sites by measuring the migration of DNA from immobilized individual cell nuclei. In this assay, the cells are embedded in agarose gel on microscopic slides, lysed and then electrophoresed under alkaline condition. Cells with damaged DNA show increased migration of DNA fragments from the nucleus and the length of the migration indicates the amount of DNA strand breakage; the latter can be estimated by manual and computerized image scoring procedures (Kumaravel et al., 2009). The technique is highly sensitive for detecting DNA damage in any eukaryotic cell type and requires only a few cells. The minimal technical requirements for doing this as say in vitro and in vivo are well established (Cotelle and Férard, 1999; Tice et al., 2000; Hartmann et al., 2003). The Comet assay is sufficiently sensitive for detecting DNA damage in frogs (Dhawan et al., 2009).

The main aim of this work was to assess the usefulness of the Comet assay for detecting DNA damage in E. johnstonei. The efficiency of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells was also assessed using a DNA diffusion assay. Since positive Comet results do not necessarily reflect genotoxicity because DNA strand breakage may be associated with cellular apoptosis and necrosis, we used the DNA diffusion assay (Singh, 2000a) to determine the percentage of DNA strand breakage associated with apoptosis and necrosis (%NAp/N) and thereby estimate the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitroquinoline-1-oxide (4NQO) was examined based on DNA strand breakage detected with the Comet assay (Baohong et al., 2005; Kumaravel and Jha, 2006); the reproducibility of the assay in this species was also addressed.

Eleutherodactylus johnstonei specimens were captured at several sites in the Bucaramanga metropolitan area (Santander, Colombia). Research and collecting permission was given by the Corporación Regional para la Defensa de la Meseta de Bucaramanga (File PC-0014-2008, Resolution 001368). Specimen sex was determined based on species sexual dimorphism and male calling. The frogs were maintained in glass terrariums at 24 ± 2 ºC on a 12 h light/dark photoperiod, in conditions (vegetation, air circulation, humidity, illumination, etc.) that simulated the wild habitat. The frogs were fed flies, crickets, spiders, ants and mosquitoes that were captured on the campus of the Universidad Industrial de Santander (Bucaramanga, Colombia).

Blood sampling, cell counts and exposure to mutagens

Blood obtained by cardiac puncture of cold-anesthetized frogs was collected in heparinized Eppendorf tubes and placed on ice until assayed. Blood cells were counted in a Neubauer counting chamber and then diluted in 0.9% (w/v) NaCl solution to a cell density of 3.55 x 106 cells/mL. Aliquots of blood cells were treated with BLM (0.6-152.0 µg/mL) or 4NQO (1.9-60.0 µM) for 2, 4, 6, 10 and 12 h (selected on the basis of preliminary experiments). Treatments were done at 6 ± 2 ºC to minimize basal DNA strand breakage. A negative control (0.9% NaCl solution) was always included in each assay. The experiments were done at least three times.

Estimation of DNA strand breakage in E. johnstonei blood cells

DNA strand breakage in E. johnstonei blood cells was initially assayed by using the alkaline Comet assay, as described by Singh et al. (1988) but with silver staining. Subsequently, DNA strand breakage was detected by the Comet assay as follows: E. johnstonei blood cells were centrifuged (10,000 rpm) and the pellet suspended in proteinase K solution (20 µL) prepared in PK buffer (50 mM Tris-HCl, 10 mM CaCl2,pH8)at40 !g/mL (concentration determined empirically). Aliquots (20 µL) of the cell suspension were mixed with 75 !L of 1% low melting point agarose and the mixture spread on slides containing a layer of 1.3% molecular grade agarose. The slides were covered with coverslips and incubated at 6 ± 2 ºC for the agarose to solidify. After enzymatic lysis and agarose polymerization, the coverslips were removed and the slides were placed in a Comet assay tank (Cleaver Scientific Ltd, UK) containing cold alkaline electrophoresis buffer (0.3 N NaOH, 1 mM EDTA, pH 13) for 25 min. Electrophoresis was done at 25 V and current adjusted to 300 mA. The slides were routinely exposed to this current in the dark at 6 ± 2ºCfor 30 min. After electrophoresis, the slides were placed in a staining tray and covered with a neutralizing buffer (0.4 M Tris-HCl, pH 7.5) in the dark for 5 min. Silver staining was done as indicated by Díaz et al. (2009).

DNA damage was expressed as arbitrary units based on the classification of comets into five categories (0-4) proposed by Collins et al. (1997). The total amount of DNA strand breakage was expressed in total arbitrary units (AUT) defined as: AUT =N0 x0+N1 x1+N2 x2+N3 x3+ N4 x 4, where Ni is the number of nuclei scored in each category (Collins, 2002). One hundred cells per slide and two slides per blood sample were analyzed using a Nikon Eclipse E200 microscope and the results from at least three independent experiments were averaged to obtain the AUT for each treatment.

For the DNA diffusion assay (Singh, 2000a), the cells were processed in a similar manner to the Comet assay, except that the nuclei were not subjected to electrophoresis. Nuclei with a diameter > 3 times the mean nuclear diameter were considered apoptotic/necrotic (Nigro et al., 2002). The total number of nuclei and the number of apoptotic/necrotic nuclei in each field were counted (minimum of 100 fields per slide) and the latter then expressed as a percentage of the former. As in the Comet assay, two slides per blood sample were analyzed and the results of at least three independent experiments were averaged to obtain the percentage of apoptotic/necrotic nuclei (%NAp/N) for each experiment.

Based on the AUT and %NAp/N estimates, the proportion of remaining DNA strand breakages was calculated (in arbitrary units) as:

where AUR corresponds to non-apoptotic/necrotic DNA strand breakages.

Statistical analysis

In all cases, the data passed the Kolmogorov-Smirnov and F-maximum tests for normality and variance homogeneity, respectively, so that parametric tests were used in subsequent data analyses. When a significant F-value was obtained in one-way analysis of variance (ANOVA) the groups were subsequently compared with Students t-test. Product-moment (Pearson) correlation analysis was used to examine the relationship between mutagen doses and the average estimates of DNA damage. A value of p < 0.05 indicated significance. All statistical analyses were done with STATISTICA V.6 software (StatSoft Inc).

Results

Cellular lysis assays

In the present work, we used the Comet assay described by Singh et al. (1988) to detect DNA strand breakage in E. johnstonei blood cells. These cells were not lysed by alkaline conditions and naked nuclei were not obtained. Although cells varied in their sensitivity to silver staining (Figure 1), neither the pH (between 10 and 13) of the incubation solution nor the length of incubation (2 h and 24 h) had any effect on the susceptibility to lysis (data not shown).

Table 1 shows the DNA strand breakage induced by BLM and 4NQO in E. johnstonei blood cells at different doses and incubation times. A marked dose-response relationship was observed for both doses and incubation times. Dose-response curves to BLM and 4NQO after a 12 h exposure (Table 2) showed marked correlations (R = 0.83 and 0.90, p 0.05; respectively). DNA strand breakage increased significantly (< 0.05) from a concentration of 4.7 µg/mL of BLM and 1.9 !M of 4NQO. The %NAp/N in E. johnstonei ranged from 0% to 8% (mean: 2.8%). The assay reproducibility under the conditions in this model was con sistently high, with coefficients of variation < 10%.

Discussion

In this work, we used a modified Comet assay to detect DNA strand breakage in the direct-developing frog E. johnstonei. Previous studies of DNA damage in frogs used alkaline treatment to lyse the cells prior to analysis by the Comet assay (Table 3). Alkaline conditions are generally sufficient to cause cellular lysis in all frog species.

The intactness of sperm DNA is regularly analyzed with the Comet assay after alkaline and enzymatic treatments (Speit et al., 2009). However, alkaline/enzymatic treatment was particularly aggressive to nuclear stability in E. johnstonei blood cells. For this reason, we used a neutral (pH 8) and single enzymatic digestion with proteinase K in situ in agarose gels; this procedure considerably reduced the assay costs and time. The temperature during cellular lysis is another critical variable that affects basal DNA damage in the Comet assay, as indicated in previous reports (Speit et al., 1999; Banáth et al., 2001). In E. johnstonei erythrocytes, enzymatic lysis at low temperature (6 ± 2 ºC) was ideal for obtaining naked nuclei with low levels of basal DNA damage.

The results described here show that the Comet assay can provide a good estimation of DNA damage in E. johnstonei. The assay was reproducible and sensitive enough to detect DNA strand breakage in E. johnstonei blood cells. The basal DNA damage estimated for the species agreed with previously reported values (Collins et al., 1997). In addition, the DNA damage observed here was poorly associated to apoptosis and necrosis, in contrast to the situation in humans (Tice et al., 2000), sea lions (El-Zein et al., 2006) and dolphins (Díaz et al., 2009).

This study has shown the usefulness of amphibians as bio-indicators. A simultaneous study (Meza-Joya et al.,in preparation) in our laboratory examined the toxic and genotoxic effects of a glyphosate-based herbicide (Roundup®SL -Cosmoflux®411F) on E. johnstonei. The study again showed that the Comet assay was highly sensitive for detecting DNA damage induced by this herbicide. This finding suggests that the Comet assay is an accurate method for detecting DNA damage in E. johnstonei after exposure to environmental xenobiotics.

In conclusion, the alkaline Comet assay (Singh et al., 1988) was inappropriate for measuring DNA strand breakage in E. johnstonei. Alkaline lysis can be replaced by enzymatic lysis (proteinase K), with good results. In contrast, combined alkaline/enzymatic treatment or long incubations (overnight) at 37 ºC with proteinase K generate unstable nuclei and result in consistently elevated basal DNA damage. The contribution of apoptosis and necrosis to the overall DNA damage in E. johnstonei was negligible, as assessed by the Comet assay. The Comet assay is a repro ducible, sensitive method for detecting DNA strand breakage in E. johnstonei.

Acknowledgments

The Corporación Regional para la Defensa de la Meseta de Bucaramanga (CDMB) provided the research and collecting permits. This work was supported by the Vicerrectoría de Investigaciones y Extensión, Universidad Industrial de Santander, Colombia (Grant No. 5163).

Associate Editor: Daisy Maria F. Salvadori License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.